早期髓系祖细胞免疫抑制功能的分析
发布时间：2019-06-21 15:34
【摘要】：目的：髓源性抑制细胞(Myeloid-derived suppressor cells,MDSCs)为未成熟但具免疫抑制能力的髓系细胞。MDSCs与肿瘤等疾病的发生密切相关,是免疫学领域近年来的热点之一。MDSCs在细胞构成上具有高度异质性,但迄今的研究局限于单核细胞样和粒细胞样MDSCs两个亚群。MDSCs是否还包括其它细胞群尤其是髓系发生早期的细胞一直是MDSC研究亟待解决的关键问题之一。据报道,小鼠胚胎干细胞和造血干细胞在体外可产生一种在分化潜能上相当于粒细胞/巨噬细胞祖细胞(GMPs)但呈现抑制活性的细胞群,提示体内GMPs可能具有免疫调节功能。本研究的目的是,通过系统地检测髓系分化通路早期系列细胞群对T细胞的抑制能力,探明髓系祖细胞的免疫调节功能属性并界定MDSC的发育起源。方法：1)小鼠Lewis肺癌细胞模型构建：接种对数生长期的LLC细胞于6-8周龄近交系C57BL/6J小鼠左腋皮下；4周后选择所长肿瘤大小适宜(直径约1.5cm)的动物用于实验。2)骨髓细胞分离与制备：取胫骨与股骨骨髓,去除红细胞,制备成骨髓单细胞悬液。3)细胞表型分析与分选：细胞用抗标志蛋白抗体(与荧光素分子结合)标记；通过流式细胞分析仪分析细胞表型和频率,通过流式细胞分选仪分选纯化特定细胞群。4)非特异性T细胞抑制实验：实验设置三个组别即待测细胞组、非免疫细胞(使用纯化的巨核细胞/红细胞祖细胞,MEP)对照组和空白对照组(以培液代替骨髓细胞)；骨髓细胞分别与经CFSE染色的C57BL/6J小鼠脾脏细胞在抗CD3/抗CD28抗体存在下共培养72小时；流式术测定CFSE荧光强度变化。5)特异性T细胞抑制实验：实验设计及方法与非特异性T细胞抑制实验类似,不同之处是以经丝裂霉素c处理的BABL/c小鼠脾脏细胞(作为抗原提呈细胞)代替抗CD3/抗CD28抗体刺激C57BL/6J小鼠脾脏T细胞。6)统计方法：数据统计采用SPSS17.0统计软件进行独立样品T检验分析。统计结果以平均值±标准差(standard deviation, SD)表示。结果：1)粒细胞/巨噬细胞祖细胞(GMPs)表现极强的免疫抑制能力。GMPs是MDSCs两个亚群的前体细胞。本课题首先分析了GMPs对非特异和抗原特异性激活的T细胞增殖的影响。在非特异T细胞反应中,分离自荷瘤小鼠的GMPs (T-GMP,T:tumor)呈现极强的T细胞抑制活性。加入T-GMP后,T细胞的增殖指数为0.82±0.45%(分裂T细胞/总脾脏细胞百分比,三次独立实验,下同),显著低于荷瘤小鼠MEP(T-MEP)添加组的22.40±2.09%(P0.01)和无骨髓细胞添加组(Medium)的(27.87±3.44%)(P0.001)。后两者间则无明显差异,亦即非免疫细胞MEP对T细胞增殖无抑制作用。出乎意料的是,分离自正常小鼠(注射PBS)的GMP (N-GMP, N:normal)也能有效地抑制T细胞增殖并表现出与T-GMP相当的抑制活性。N-GMP和N-MEP组T细胞的增殖指数分别为1.17±1.03%和23.10±2.45%(P0.01)。在特异性T细胞反应抑制实验中,我们得到了类似的结果。T-GMP组、T-MEP组和Medium组的T细胞增殖指数分别为0.32±0.08%、3.15±0.52%和4.37士0.9%(0.32±0.08% VS 3.15±0.52%,P0.05；0.32±0.08%VS 4.37±0.9%,P0.01)。N-GMP组和N-MEP组的T细胞增殖指数分别为0.36±0.02%和3.76±0.63%(P0.01)。2)共同髓系祖细胞(CMPs)是髓系发生通路中最早的抑制细胞群。接下来检测了GMPs上游的系列细胞群,包括共同髓系祖细胞(CMPs)、多能祖细胞(MPPs)和造血干细胞(HSCs)。结果显示,分离自荷瘤小鼠CMP(T-CMP)和正常小鼠CMP (N-CMP)均能有效地抑制T细胞的增殖。在非特异T细胞反应中,T-CMP和N-CMP两组T细胞的增殖指数分别为4.51±1.89%和5.21±2.91%,而相对应的T-MEP组和N-MEP组的T细胞增殖指数分别为26.70±2.09%和28.00±2.45%。在特异性T细胞反应中,T-CMP组和N-CMP组T细胞的增殖指数分别为1.36±0.15%和1.47±0.21%,而相对应的T-MEP组和N-MEP组的T细胞增殖指数分别为3.15±0.52%和3.76±0.63%。但是,无论是分离自正常小鼠还是荷瘤小鼠的MPPs和HSCs,均不表现T细胞抑制作用。3)早期髓系祖细胞具有比其下游细胞更强的抑制活性。我们进而比较了GMPs和CMPs与经典MDSCs的抑制能力。在相同的细胞比例(GMP或MDSC:脾脏细胞=1：2)下,GMP抑制能力明显强于MDSCs(T-GMP VST-MDSC: 0.82±0.45%VS 7.61±1.51%, P0.05; N-GMP VS N-MDSC:1.17±1.03% VS 11.50±1.05%,P0.001)。来源于生理与肿瘤鼠的CMP(CMP:脾脏细胞=1：8)的免疫抑制能力同样强于MDSCs (MDSC:脾脏细胞=1：8)(5.21±2.91 VS 28.00±.45%,P0.01,正常小鼠；4.51±1.89%VS 26.7±2.09%,P0.01,荷瘤小鼠)。4)骨髓造血干/祖细胞在肿瘤进程中呈现特征性的发育变化。最后,测定了造血干/祖细胞群在肿瘤状态下丰度的变化。在荷瘤小鼠中,GMP占骨髓细胞的比例为1.120±0.071%,显著高于生理状态下的GMP含量(0.649±0.058%,三次以上独立实验,P0.01)。肿瘤CMP含量则低于生理状态下的含量(0.111±0.019% VS0.233±0.032%,P0.01)。在生理和肿瘤状态下,MPP的含量分别为0.033±0.003%和0.199±0.040%(P0.05),HSC的含量分别为0.033±0.007%和0.176±0.0136%(P0.01)。结论：1)髓系祖细胞GMPs和CMPs具有免疫抑制功能,并表现比其下游细胞(经典MDSCs)更强的抑制活性。这些发现不仅让我们鉴定出了髓源性抑制细胞家族的两个新成员并确定CMPs是髓系分化通路中最早出现的抑制性细胞群,而且折示它们在骨髓组织自稳态维护以及防止自身免疫和过度免疫反应中可能起重要作用。2)髓系细胞(包括CMPs、GMPs和经典MDSCs)的免疫抑制功能是其内在功能特性,与病理状态无关；但是,肿瘤环境可以刺激HSC增殖并促进其向髓系细胞尤其GMPs的分化。这些结果表明髓系抑制细胞是机体免疫调节网络的固有组成部分,同时为其在肿瘤等病态下扩增的机理提供了新的解释视角。
[Abstract]:Objective: Myeloid-derived support cells (MDSCs) are immature but immunosuppressed myeloid cells. MDSCs are closely related to the occurrence of diseases such as tumors, and is one of the hot spots in the field of immunology. MDSCs are highly heterogeneous in cell formation, but studies to date are limited to both monocyte and granulocyte-like MDSCs. Whether MDSCs also include other cell populations, especially the early-stage cells of the myeloid lineage, have been one of the key issues to be addressed in the MDSC study. It has been reported that mouse embryonic stem cells and hematopoietic stem cells can in vitro produce a cell population that is equivalent to granulocyte/ macrophage progenitor cells (GMPs) in differentiation potential but exhibit inhibitory activity, suggesting that GMPs in vivo may have an immunomodulatory function. The purpose of this study was to investigate the ability of the early series of cell populations to inhibit T cells in the early series of myeloid differentiation and to explore the functional properties of myeloid progenitor cells and to define the development origin of MDSCs. Methods:1) The mouse Lewis lung cancer cell model was constructed: the LLC cells in the logarithmic growth phase were subcutaneously implanted in the left axilla of the C57BL/ 6J mice at 6-8 weeks of age; after 4 weeks, the animals with the appropriate tumor size (about 1.5 cm in diameter) were selected for the experiment.2) The bone marrow cells were isolated and prepared: taking the tibia and the femur bone marrow, removing red blood cells to prepare a single cell suspension of bone marrow;3) cell phenotypic analysis and sorting: labeling the cell with an anti-marker protein antibody (in combination with a fluorescein molecule); analyzing the cell phenotype and frequency by flow cytometry; and sorting and purifying a specific cell group by flow cytometry.4) the non-specific T cell inhibition experiment: three groups of cells to be tested, non-immune cells (using purified megakaryocyte/ erythrocyte progenitor cells, MEPs) control group and blank control group (replacing bone marrow cells with culture liquid); bone marrow cells were co-cultured with CFSE-stained C57BL/ 6J mouse spleen cells for 72 hours in the presence of anti-CD3/ anti-CD28 antibody; flow cytometry was used to determine the change of CFSE fluorescence intensity. The experimental design and method were similar to those of non-specific T cell inhibition, except that the spleen cells of the BABL/ c mice treated with mitomycin c (as antigen-presenting cells) were used in place of the anti-CD3/ anti-CD28 antibody to stimulate the spleen T cells of C57BL/ 6J mice. The data statistics are analyzed by using the SPSS17.0 statistical software for independent sample T test. The statistical results are represented by standard deviation (SD). Results:1) The granulocyte/ macrophage progenitor cells (GMPs) showed strong immunosuppression ability. GMPs are the precursor cells of two subpopulations of MDSCs. The effects of GMPs on the proliferation of non-specific and antigen-specific activated T cells were analyzed. In the non-specific T cell reaction, the GMPs (T-GMP, T: tumor) isolated from the tumor-bearing mice showed strong T-cell inhibitory activity. After the addition of T-GMP, the proliferation index of T-cells was 0.82-0.45% (percentage of divided T-cells/ total spleen cells and three independent experiments, the same below), which was significantly lower than that of the control group (22.40-2.09% (P0.01) and (27.87-3.44%) of the non-bone marrow cell-added group (P0.001). There was no significant difference between the two groups, that is, the non-immune cell MEP had no inhibitory effect on T cell proliferation. Surprisingly, GMP (N-GMP, N: normal) isolated from normal mice (injected PBS) can also effectively inhibit T cell proliferation and exhibit a comparable inhibitory activity as T-GMP. The proliferation index of T cells in N-GMP and N-MEP group was 1.17, 1.03% and 23.10-2.45%, respectively (P0.01). In the specific T cell reaction inhibition assay, similar results were obtained. The T-cell proliferation index of T-GMP group, T-MEP group and medium group was 0.32-0.08%, 3.15-0.52% and 4.37-0.9% (0.32-0.08% VS 3.15-0.52%, P0.05; 0.32-0.08% VS 4.37-0.9%, P0.01). The T-cell proliferation index of N-GMP group and N-MEP group was 0.36-0.02% and 3.76-0.63% (P0.01). The series of cell populations upstream of GMPs were then tested, including common myeloid progenitor cells (CMPs), pluripotent progenitor cells (MPPs) and hematopoietic stem cells (HSCs). The results showed that both CMP (T-CMP) and normal mouse CMP (N-CMP) were effective in the inhibition of T cell proliferation. In the non-specific T cell reaction, the proliferation index of T cells in T-CMP and N-CMP was 4.51% 1.89% and 5.21% 2.91%, respectively, while the corresponding T-MEP group and N-MEP group had a T-cell proliferation index of 26.70% 2.09% and 28.00-2.45%, respectively. In the specific T-cell reaction, the proliferation index of T-and N-group T cells was 1.36-0.15% and 1.47-0.21%, respectively, while the corresponding T-MEP group and N-MEP group had a T-cell proliferation index of 3.15-0.52% and 3.76-0.63%, respectively. However, neither the MPPs nor the HSCs isolated from the normal mice or the tumor-bearing mice did not show a T-cell inhibition.3) The early myeloid progenitor cells had a stronger inhibitory activity than the downstream cells. In turn, we compared the ability of GMPs and CMPs to suppress the classical MDSCs. Under the same ratio of cells (GMP or MDSC: spleen cells = 1:2), the inhibition of GMP was significantly stronger than that of MDSCs (T-GMP VST-MDSC: 0.82, 0.45% VS 7.61, 1.51%, P0.05; N-GMP VS N-MDSC: 1.17, 1.03% VS 11.50, 1.05%, P0.001). The immunosuppression ability of CMP (CMP: spleen cells = 1:8) derived from physiological and tumor mice was also stronger than that of MDSCs (MDSC: spleen cells = 1:8) (5.21% 2.91 VS 28.00).45%, P 0.01, normal mice; 4.51% 1.89% VS 26.7% 2.09%, P 0.01, Tumor-bearing mice).4) The bone marrow hematopoietic stem/ progenitor cells present a characteristic development change in the tumor process. Finally, the changes of the abundance of the hematopoietic stem/ progenitor cell population in the tumor were determined. In the tumor-bearing mice, the proportion of GMP in the bone marrow cells was 1.120-0.071%, which was significantly higher than that in the physiological state (0.649-0.058%, more than three independent experiments, P0.01). The content of CMP was lower than that in physiological state (0.111-0.019% VS.233-0.032%, P0.01). In the physiological and tumor state, the content of MPP was 0.033-0.003% and 0.199-0.040% (P0.05). The content of HSC was 0.033-0.007% and 0.176-0.0136%, respectively (P0.01). Conclusion:1) The myeloid progenitor cells GMPs and CMPs have an immunosuppressive function and show stronger inhibitory activity than its downstream cells (classical MDSCs). These findings not only allowed us to identify two new members of the myeloid-derived suppressor cell family and to determine that the CMPs were the earliest inhibitory cell populations in the myeloid differentiation pathway, and it is shown that they may play an important role in the self-stable maintenance of the bone marrow tissue and the prevention of autoimmune and hyperimmune responses.2) The immunosuppressive function of myeloid cells (including CMPs, GMPs and the classical MDSCs) is its intrinsic function, not related to the pathological state; however, The tumor environment can stimulate the proliferation of HSC and promote its differentiation into the myeloid cells, especially GMPs. These results show that the myeloid-inhibiting cell is an integral part of the immunomodulatory network of the organism, and provides a new interpretation view for the mechanism of its amplification in the condition of tumor.
【学位授予单位】：广西师范大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R392
本文编号：2504183
[Abstract]:Objective: Myeloid-derived support cells (MDSCs) are immature but immunosuppressed myeloid cells. MDSCs are closely related to the occurrence of diseases such as tumors, and is one of the hot spots in the field of immunology. MDSCs are highly heterogeneous in cell formation, but studies to date are limited to both monocyte and granulocyte-like MDSCs. Whether MDSCs also include other cell populations, especially the early-stage cells of the myeloid lineage, have been one of the key issues to be addressed in the MDSC study. It has been reported that mouse embryonic stem cells and hematopoietic stem cells can in vitro produce a cell population that is equivalent to granulocyte/ macrophage progenitor cells (GMPs) in differentiation potential but exhibit inhibitory activity, suggesting that GMPs in vivo may have an immunomodulatory function. The purpose of this study was to investigate the ability of the early series of cell populations to inhibit T cells in the early series of myeloid differentiation and to explore the functional properties of myeloid progenitor cells and to define the development origin of MDSCs. Methods:1) The mouse Lewis lung cancer cell model was constructed: the LLC cells in the logarithmic growth phase were subcutaneously implanted in the left axilla of the C57BL/ 6J mice at 6-8 weeks of age; after 4 weeks, the animals with the appropriate tumor size (about 1.5 cm in diameter) were selected for the experiment.2) The bone marrow cells were isolated and prepared: taking the tibia and the femur bone marrow, removing red blood cells to prepare a single cell suspension of bone marrow;3) cell phenotypic analysis and sorting: labeling the cell with an anti-marker protein antibody (in combination with a fluorescein molecule); analyzing the cell phenotype and frequency by flow cytometry; and sorting and purifying a specific cell group by flow cytometry.4) the non-specific T cell inhibition experiment: three groups of cells to be tested, non-immune cells (using purified megakaryocyte/ erythrocyte progenitor cells, MEPs) control group and blank control group (replacing bone marrow cells with culture liquid); bone marrow cells were co-cultured with CFSE-stained C57BL/ 6J mouse spleen cells for 72 hours in the presence of anti-CD3/ anti-CD28 antibody; flow cytometry was used to determine the change of CFSE fluorescence intensity. The experimental design and method were similar to those of non-specific T cell inhibition, except that the spleen cells of the BABL/ c mice treated with mitomycin c (as antigen-presenting cells) were used in place of the anti-CD3/ anti-CD28 antibody to stimulate the spleen T cells of C57BL/ 6J mice. The data statistics are analyzed by using the SPSS17.0 statistical software for independent sample T test. The statistical results are represented by standard deviation (SD). Results:1) The granulocyte/ macrophage progenitor cells (GMPs) showed strong immunosuppression ability. GMPs are the precursor cells of two subpopulations of MDSCs. The effects of GMPs on the proliferation of non-specific and antigen-specific activated T cells were analyzed. In the non-specific T cell reaction, the GMPs (T-GMP, T: tumor) isolated from the tumor-bearing mice showed strong T-cell inhibitory activity. After the addition of T-GMP, the proliferation index of T-cells was 0.82-0.45% (percentage of divided T-cells/ total spleen cells and three independent experiments, the same below), which was significantly lower than that of the control group (22.40-2.09% (P0.01) and (27.87-3.44%) of the non-bone marrow cell-added group (P0.001). There was no significant difference between the two groups, that is, the non-immune cell MEP had no inhibitory effect on T cell proliferation. Surprisingly, GMP (N-GMP, N: normal) isolated from normal mice (injected PBS) can also effectively inhibit T cell proliferation and exhibit a comparable inhibitory activity as T-GMP. The proliferation index of T cells in N-GMP and N-MEP group was 1.17, 1.03% and 23.10-2.45%, respectively (P0.01). In the specific T cell reaction inhibition assay, similar results were obtained. The T-cell proliferation index of T-GMP group, T-MEP group and medium group was 0.32-0.08%, 3.15-0.52% and 4.37-0.9% (0.32-0.08% VS 3.15-0.52%, P0.05; 0.32-0.08% VS 4.37-0.9%, P0.01). The T-cell proliferation index of N-GMP group and N-MEP group was 0.36-0.02% and 3.76-0.63% (P0.01). The series of cell populations upstream of GMPs were then tested, including common myeloid progenitor cells (CMPs), pluripotent progenitor cells (MPPs) and hematopoietic stem cells (HSCs). The results showed that both CMP (T-CMP) and normal mouse CMP (N-CMP) were effective in the inhibition of T cell proliferation. In the non-specific T cell reaction, the proliferation index of T cells in T-CMP and N-CMP was 4.51% 1.89% and 5.21% 2.91%, respectively, while the corresponding T-MEP group and N-MEP group had a T-cell proliferation index of 26.70% 2.09% and 28.00-2.45%, respectively. In the specific T-cell reaction, the proliferation index of T-and N-group T cells was 1.36-0.15% and 1.47-0.21%, respectively, while the corresponding T-MEP group and N-MEP group had a T-cell proliferation index of 3.15-0.52% and 3.76-0.63%, respectively. However, neither the MPPs nor the HSCs isolated from the normal mice or the tumor-bearing mice did not show a T-cell inhibition.3) The early myeloid progenitor cells had a stronger inhibitory activity than the downstream cells. In turn, we compared the ability of GMPs and CMPs to suppress the classical MDSCs. Under the same ratio of cells (GMP or MDSC: spleen cells = 1:2), the inhibition of GMP was significantly stronger than that of MDSCs (T-GMP VST-MDSC: 0.82, 0.45% VS 7.61, 1.51%, P0.05; N-GMP VS N-MDSC: 1.17, 1.03% VS 11.50, 1.05%, P0.001). The immunosuppression ability of CMP (CMP: spleen cells = 1:8) derived from physiological and tumor mice was also stronger than that of MDSCs (MDSC: spleen cells = 1:8) (5.21% 2.91 VS 28.00).45%, P 0.01, normal mice; 4.51% 1.89% VS 26.7% 2.09%, P 0.01, Tumor-bearing mice).4) The bone marrow hematopoietic stem/ progenitor cells present a characteristic development change in the tumor process. Finally, the changes of the abundance of the hematopoietic stem/ progenitor cell population in the tumor were determined. In the tumor-bearing mice, the proportion of GMP in the bone marrow cells was 1.120-0.071%, which was significantly higher than that in the physiological state (0.649-0.058%, more than three independent experiments, P0.01). The content of CMP was lower than that in physiological state (0.111-0.019% VS.233-0.032%, P0.01). In the physiological and tumor state, the content of MPP was 0.033-0.003% and 0.199-0.040% (P0.05). The content of HSC was 0.033-0.007% and 0.176-0.0136%, respectively (P0.01). Conclusion:1) The myeloid progenitor cells GMPs and CMPs have an immunosuppressive function and show stronger inhibitory activity than its downstream cells (classical MDSCs). These findings not only allowed us to identify two new members of the myeloid-derived suppressor cell family and to determine that the CMPs were the earliest inhibitory cell populations in the myeloid differentiation pathway, and it is shown that they may play an important role in the self-stable maintenance of the bone marrow tissue and the prevention of autoimmune and hyperimmune responses.2) The immunosuppressive function of myeloid cells (including CMPs, GMPs and the classical MDSCs) is its intrinsic function, not related to the pathological state; however, The tumor environment can stimulate the proliferation of HSC and promote its differentiation into the myeloid cells, especially GMPs. These results show that the myeloid-inhibiting cell is an integral part of the immunomodulatory network of the organism, and provides a new interpretation view for the mechanism of its amplification in the condition of tumor.
【学位授予单位】：广西师范大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R392
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